This proposal aims to use a novel approach to enable and develop a validated high throughput cell-based screen to identify small molecule modulators of the Galanin 3 receptor (GAL3R). The Gal3R belongs to the G protein-coupled receptor family of proteins and together with two other closely related receptors GAL1R and GAL2R and their endogenous ligand galanin, are involved in a variety of physiological and pathophysiological processes. The GAL3R in particular has been strongly implicated in addiction and mood related disorders such as anxiety and depression. It has been the target of many drug discovery programs within the pharmaceutical industry but despite the significant resources and effort devoted to discovery of galanin receptor subtype selective small molecule modulators, there have been very few reports for the discovery of such molecules and in addition, all access to primary screening data remains proprietary information. The GAL3R has proven difficult to enable in cell-based functional assays due to its apparent poor cell surface expression in recombinant systems. This application proposes to use a modified GAL3R that facilitates its cell surface expression while maintaining wildtype GAL3R pharmacology. The modified GAL3R will be used to develop a primary cell-based cAMP biosensor assay to detect selective agonists, potentiators, and antagonists of GAL3R all in a single screen. Following assay optimization of the screening conditions, the performance of the assays will be evaluated by challenging them against a test library of small molecules with known pharmacological activities (Sigma-LOPAC). Once validated, the primary assay will be submitted to the Molecular Libraries Screening Center Network (MLSCN) high throughput screening resources with the aim of identifying novel molecular probes that modulate GAL3R activity. Hits from this campaign will be counterscreened against the parental cell line used to generate the GAL3R cell line and two other cell lines expressing either GAL1R or GAL2R. Confirmed and GAL3R selective hits will be interrogated in an orthoganol cell-based functional assay to further characterize their pharmacological properties. Combined, these approaches will result in the identification of GAL3R selective modulators (chemical probes) which will facilitate dissection of the biological role that GAL3R plays in normal physiological processes as well as in disease states. PUBLIC HEALTH RELEVANCE: The Galanin 3 receptor (GAL3R) is expressed in the periphery and has limited expression in the CNS. It has been demonstrated to play a role in neurological disorders such as addiction and depression. This proposal seeks to develop a high throughput cell-based functional assay to identify GAL3R selective ligands that can be used as molecular probes to interrogate GAL3R receptor function in normal physiological processes and disease states.